Various synthetic substrates for .gamma.-GTP activity determination have been proposed. Widely employed substrates among them are L-.gamma.-glutamyl-p-nitroanilide or a salt thereof and L-.gamma.-glutamyl-3-carboxy-4-nitroanilide or a salt thereof which generate p-nitroaniline or 3-carboxy-4-nitroaniline (i.e., 5-amino-2-nitrobenzoic acid), respectively, by the enzymatic action of .gamma.-GTP. For determining .gamma.-GTP, the thus generated product is determined by spectrophotometry.
However, L-.gamma.-glutamyl-p-nitroanilide or its salt, when dissolved in a buffer solution for .gamma.-GTP activity determination, is precipitated as crystals over time due to poor solubility. Additionally, it undergoes non-enzymatic decomposition (hydrolysis) and releases p-nitroaniline when stored over a period of time. On the other hand, L-.gamma.-glutamyl-3-carboxyl-4-nitroanilide or its salt, while having a high solubility, is also non-enzymatically decomposed (hydrolyzed) to release 3-carboxy-4-nitroaniline during storage.
Various studies have been conducted in order to overcome these problems. With respect to L-.gamma.-glutamyl-p-nitroanilide, for example, it has been proposed to improve solubility by the addition of an acid, an organic solvent, a surfactant, etc. (see Clinica Chimica Acta, Vol. 65, p. 21-27 (1975)). Addition of cyclodextrin (see JP-A-57-74099 corresponding to WO-8201564 (U.S., DE), the term "JP-A" as used herein means an "unexamined published Japanese patent application") or a crown ether (see JP-A-60-16599) is suggested for improvement of solubility and inhibition of non-enzymatic decomposition (hydrolysis). Nevertheless, a technique for satisfying both solubility and stability of this substrate has not yet been developed.
Also with reference to L-.gamma.-glutamyl-3-carboxy-4-nitroanilide, no effective means to inhibit non-enzymatic decomposition (hydrolysis) has been reported. Accordingly, because of the difficulty in supplying L-.gamma.-glutamyl-3-carboxyl-4-nitroanilide in solution, the substrate must be supplied as a freeze-dried product or as a powder package to be dissolved in a buffer solution at the time of use. Moreover, since the pot life of the thus prepared substrate solution is only about 1 month even in a refrigerator (2.degree. to 8.degree. C.) due to non-enzymatic decomposition (hydrolysis), the clinical technician must prepare fresh substrate solution as needed and often each time an assay is conducted.
Inhibition of L-.gamma.-glutamyl-3-carboxy-4-nitroanilide from non-enzymatic decomposition (hydrolysis) would make it possible to provide a solution-type reagent free from precipitation thereby eliminating the need for preparing fresh solution each time an assay is conducted.